The present invention relates to a novel plasmid, which we have named "pSAN 181", and to a process for extracting the plasmid from the mycelia of the species Streptomyces fulvoviridis.
Plasmids are autonomously replicating, normally cyclic, double-stranded DNA molecules which do not normally form part of the cellular chromosome and which are believed to carry genetic information which is not essential for the growth of the host cell, whilst the chromosome carries all of the essential genetic information. The plasmids are described as "normally cyclic" as this is the form that they may be seen to adopt in isolation under laboratory conditions; the form adopted under other conditions may or may not be different. The functions in nature of certain plasmids have been discovered, for example, some are known to code for apparatus (e.g. enzymes) providing the host cell with resistance to antibiotics, but many plasmids are "cryptic", i.e. their function in the cell has not been elucidated.
In the field of genetic engineering, however, plasmids are invaluable, in that they provide three different, but related, functions. First they provide a mechanism whereby a selected DNA molecule (generally coding for some valuable function, such as the production of proinsulin or interferon) may be multiplied many times by natural biological activity; this is called "cloning". Second, they provide a mechanism whereby the activity of that DNA molecule may be expressed, i.e. a cell may be induced to produce the material coded for by that DNA molecule by incorporating the genetic information on the plasmid (which includes the added DNA molecule coding for the desired material) into a living cell. Third, they act as transportation vectors to move DNA from one genome to another.
The insertion of a DNA molecule containing desired genetic information into a plasmid is effected by cleaving the plasmid and then splicing the length of added DNA molecule onto each of the free ends of the cleaved plasmid, to reform a larger cyclic DNA molecule, i.e. a larger plasmid. This cleavage is effected by means of an enzyme known as a restriction endonuclease, which catalyses the hydrolysis of the DNA molecule at one or more specific sites. The activity of a particular endonuclease is limited to the catalysis of hydrolysis at a characteristic site and thus causes cleavage of the plasmid at that site. Since the precise nucleotide sequence of many plasmids has not yet been elucidated, plasmids can be identified and characterised by the number and relative separation of the sites on the circular DNA molecular at which various restriction endonucleases cleave the molecule.
To maximise control of genetic engineering work with plasmids, it is desirable that any given restriction endonuclease should cleave the plasmid at a single site only; if the endonuclease cleaves the plasmid at more than one site, a variety of fragments will be formed and these may combine in many different ways. Normally, the smaller the plasmid, the greater will be the chance that a given restriction endonuclease will cleave it at one site only. Ideally, in order to give the greatest possible flexibility, the plasmid will be susceptible to attack by a number of different restriction endonucleases, each capable of cleaving the plasmid at a single characteristic site.